JP2018044935A - Chemical analysis device, pretreatment device and chemical analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chemical analysis device to quantify a detection object quickly and with high sensitivity, and a pretreatment device and a chemical analysis method for use in the chemical analysis device.SOLUTION: A chemical analysis device 600 has: a pretreatment part 60 that stores a molecule mold polymer to capture a molecule having a polar group included in a sample, and a quantification part 65 that quantifies a component included in the sample passed through the pretreatment part 60.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a chemical analysis device, a pretreatment device used in the chemical analysis device, and a chemical analysis method, and more particularly to a chemical analysis device used for detection of a biomolecule, a pretreatment device used in the chemical analysis device, and a chemical analysis method. .

  The chemical substances that should be managed in the fields of clinical examination, environment, hygiene, disaster prevention, etc. are very diverse and there are many types. For example, hormone molecules, endocrine disrupting substances, soil pollutants in factory sites, asbestos generated from building materials, chemical substances that cause off-flavors and off-flavors generated from foods and containers, or their manufacturing equipment, and the like. Many of such chemical substances are small molecules, and usually only a very small amount is contained in the measurement object. It is extremely important to detect such a trace amount of chemical substances quickly and with high sensitivity in order to ensure safety in each field.

  For example, in the life science field, there has been a movement to acquire tailor-made prevention by acquiring information that represents the current state of the body sensitively through metabolomic analysis. In order to realize such a method, it is required to analyze relatively low molecular weight metabolites such as amino acids and organic acids and apply the results to diagnosis, so mass spectrometry has become an important technology. Yes. As a mass spectrometer, for example, a biological sample such as blood, serum, urine, saliva or the like is injected into a vacuum with a high voltage, and ionized components are separated and detected to obtain a mass spectrum. It has been.

  At these measurement sites, there is a need for a measurement technique that can detect a target chemical substance with high sensitivity on the spot. Furthermore, in the measurement field, miniaturization of the measuring device is also required. In order to meet these requirements, when a specific chemical substance is selectively detected as a detection target (hereinafter also referred to as a target molecule), a substance that disturbs the detection (hereinafter referred to as a measurement inhibitory substance). ) Is important to remove efficiently.

  For example, Patent Document 1 has a pre-column unit that accommodates a carrier that removes a measurement-inhibiting substance in a sample upstream of a reaction chamber, and the reaction chamber and the pre-column unit use a centrifugal force due to rotation to sample and reagents. Is disclosed.

JP 2011-27421 A

  The reaction chip described in Patent Document 1 requires a fine structure for removing measurement-inhibiting substances in a specimen and a mechanism for generating centrifugal force, which complicates the entire structure. In addition, this reaction chip uses a centrifugal force due to rotation to send a sample or a reagent, so that it takes time to send the solution and is not suitable for high-throughput analysis.

  Moreover, in a mass spectrometer, for example, measurement inhibitory substances such as phospholipids contained in a biological sample may be preferentially ionized over target molecules. In this case, target molecules that should be detected originally are not ionized, and errors may occur in the analysis results, resulting in a decrease in analysis accuracy.

  SUMMARY OF THE INVENTION An object of the present invention is to provide a chemical analysis apparatus that rapidly and highly sensitively quantifies a detection object, a pretreatment apparatus used in the chemical analysis apparatus, and a chemical analysis method.

  A preferred embodiment of the chemical analyzer according to the present invention includes a pretreatment unit that contains a molecular template polymer that captures a molecule having a polar group contained in a sample, and the sample that is passed through the pretreatment unit. And a quantification unit for quantifying the components to be quantified.

  A preferred embodiment of the pretreatment apparatus according to the present invention is a pretreatment apparatus used in a chemical analysis apparatus that quantitatively analyzes components contained in a specimen, and the pretreatment apparatus is contained in the specimen. A molecular template polymer that captures a molecule having a polar group is provided, and the molecule having the polar group is removed from the specimen by the molecular template polymer.

  Further, as a preferred embodiment of the chemical analysis method according to the present invention, the specimen is passed through a pretreatment unit containing a molecular template polymer that captures a molecule having a polar group, and the liquid passed through the pretreatment unit. It is characterized by quantitatively analyzing components contained in a specimen.

  ADVANTAGE OF THE INVENTION According to this invention, the chemical analyzer which quantifies a detection target object rapidly and with high sensitivity, the pre-processing apparatus used for this chemical analyzer, and a chemical analysis method are realizable.

1 is a diagram showing a schematic configuration of a chemical analyzer 600 according to Example 1. FIG. It is a figure which shows typically the typical manufacturing method of a molecular template polymer. It is a figure which shows typically the cross section of the molecular template polymer fine particle 61. FIG. It is a figure which shows typically the cross section of the molecular template polymer fine particle 61. FIG. It is a processing flow figure when performing from a pretreatment process to an analysis process using a molecular template polymer. It is a figure which shows schematic structure of the pre-processing apparatus 400 which concerns on Example 2. FIG. It is a figure which shows the relationship between a phospholipid density | concentration and a light absorbency. It is a figure which shows the relationship between molecular template polymer addition concentration and phospholipid reduction concentration.

  FIG. 1 shows a schematic configuration of a chemical analyzer 600 according to the first embodiment. The chemical analyzer 600 includes a pretreatment unit 60 that accommodates the molecular template polymer fine particles 61 therein. The molecular template polymer fine particle 61 has a molecular template polymer that captures a molecule having a polar group contained in a specimen as a measurement inhibitor. The molecular template polymer captures a specific measurement inhibitor in a sample depending on the specific molecular structure of the measurement inhibitor. Capture refers to capturing by binding or interaction.

  The pretreatment unit 60 is made of a resin such as glass, PDMS, or acrylic. The molecular template polymer fine particle 61 has a function as a capturing body that captures a measurement-inhibiting substance by the molecular template polymer, and details will be described later.

  A sample introduction unit 62 that introduces a sample into the pretreatment unit 60 is connected to the pretreatment unit 60 by a flow path 621. A drain 66 is further connected to the pretreatment unit 60 by a discharge channel 661, and a fixed amount unit 65 is connected by a delivery channel 651. The discharge channel 661 and the delivery channel 651 are connected to each other by the switching unit 64 so that they can be switched.

  The quantification unit 65 quantifies the detection target (target molecule) contained in the specimen sent from the preprocessing unit 60. For example, the quantification unit 65 is a mass spectrometer, a liquid chromatography-mass spectrometer, a liquid chromatograph, a spectrophotometer. A total, a biochemical / immune automatic analyzer, etc. can be used.

  When the sample is injected from the sample introduction unit 62 into the pretreatment unit 60 through the flow path 621, the measurement inhibitor contained in the sample is captured by the molecular template polymer fine particles 61 and remains in the pretreatment unit 60. When the switching unit 64 is switched to the delivery channel 651 side, the sample solution pretreated by the pretreatment unit 60 is sent to the quantification unit 65 by the delivery channel 651 via the switching unit 64 to be detected. An object (target molecule) is quantitatively analyzed.

  As a result, it is possible to quantitatively analyze the sample solution in which the measurement inhibitory substance is significantly reduced as compared with the state before passing through the pretreatment unit 60 by the quantitative unit 65. Therefore, quantitative analysis of the target molecule can be performed quickly and with high sensitivity, and an analysis result having a high S / N (signal-to-noise ratio) can be obtained.

  The molecular template polymer fine particles 61 that have captured the measurement inhibitory substance remaining in the pretreatment unit 60 are drained by the discharge channel 661 together with unnecessary impurities by switching the switching unit 64 to the discharge channel 661 side. May be discharged from.

  In addition, the chemical analyzer of Example 1 mainly uses low molecular weight chemical substances as target molecules, but in some of the mass spectrometers for clinical tests, such low molecular weight target molecules and A component having a close molecular weight is treated as inspection noise that hinders measurement. In such a chemical analyzer, efficient concentration of target molecules and reduction of measurement-inhibiting substances determine whether test items can be implemented. In the chemical analyzer according to the first embodiment, the measurement inhibitor contained in the specimen can be efficiently removed by the pretreatment unit 60 having the molecular template polymer fine particles 61. The influence can be reduced.

  In addition, for example, a mass spectrometer injects a biological sample such as blood, serum, urine, saliva, etc. in a vacuum applied with a high voltage and causes components ionized in the vacuum to fly by an electrostatic force, thereby causing an electric action or magnetic force. Component analysis is performed in which each component is separated according to the mass-to-charge ratio by an automatic action, and the separated component is detected to obtain a mass spectrum relating to the detected intensity with respect to the mass-to-charge ratio.

  For example, when a serum component obtained by separating blood cells from blood is subjected to mass spectrometry using such a mass spectrometer, the serum component is directly injected into the mass spectrometer, or the pretreated serum component is injected into the mass spectrometer. In either case, phospholipids in serum may enter the ionization port together with the detection target (target molecule). In this case, since the phospholipid is preferentially ionized and the detection target (target molecule) that should be ionized is not ionized, an error occurs in the measurement result.

  In the chemical analyzer of the example, a measurement inhibitor having a polar group that is easily ionized is previously removed from the sample by the molecular template polymer in the pretreatment unit 60, and then the sample is passed through the quantification unit 65 for quantitative analysis. It can be performed. For this reason, when the fixed_quantity | quantitative_assay part 65 is an above-described mass spectrometer, the fall of analytical accuracy by the measurement inhibitory substance which has polar groups, such as a phospholipid, preferentially ionizing can be prevented, for example.

  In addition, when the quantification unit 65 is, for example, a liquid chromatography or spectroscopic analyzer, for example, the overlap between the peak of the detection target (target molecule) and the peak of the measurement inhibitory substance in the spectrum obtained by quantitative analysis is reduced. Can do.

  Further, when the quantification unit 65 is, for example, a biochemical / immune automatic analyzer, generation of measurement error due to adhesion of a measurement inhibitor on a substrate on which an antigen or antibody is adhered, or antigen due to a measurement inhibitor The inhibition reaction of the antibody reaction can be prevented.

  In addition, for the detection of the target molecule, in addition to the above-described mass spectrometer, the measurement unit 65 uses an electrical measurement, impedance measurement, surface plasmon resonance, a measurement unit using a crystal resonator, a spectrophotometer, or the like. Also good.

  In FIG. 1, a configuration in which one pre-processing unit 60 is provided in the quantification unit 65 is shown, but a plurality of pre-processing units 60 may be provided for one quantification unit 65. In this case, removal of the measurement inhibitory substance can be performed in parallel by the plurality of preprocessing units 60, so that more efficient and high-throughput analysis can be performed.

  As described above, a molecule that is captured by a molecular template polymer (hereinafter referred to as a molecule to be captured) is a molecule that has a polar group and is easily ionized. For example, phospholipids, phospholipid derivatives, phosphoproteins, phosphopeptides, A molecule having a phosphate group is exemplified.

  The molecular weight of the molecule to be captured is not particularly limited as long as it is a molecular weight that can be captured by the capturing body. However, since Example 1 mainly aims to detect a low molecular weight chemical substance, the molecular weight of the target molecule is approximately several. About ten to several hundred. This also applies to Example 2 below.

  FIG. 2 shows a typical production principle of the molecular template polymer (MIP) 12 included in the molecular template polymer fine particles 61. First, a recognition reaction 11 of the template molecule 10 is formed by performing a polymerization reaction in a mixture of the template molecule 10 that is the target molecule to be captured and the monomer raw material A101, monomer raw material B102, and monomer raw material C103 that interact with the template molecule 10. Let Thereafter, the molecular template polymer (MIP) 12 having the recognition site 11 can be produced by removing the template molecule 10 by washing or the like.

  The template molecule 10 is a template molecule for forming the recognition site 11, and the capture target molecule itself may be used, or a derivative or analog of the capture target molecule may be used.

  The molecular template polymer (MIP) 12 is formed using a specific template molecule 10 and can capture a chemical substance that is a target molecule for capture depending on the specific molecular structure of the target molecule. It is. The capturing body that captures the capture target molecule only needs to have at least the molecular template polymer (MIP) 12, and is made of a material other than the polymer that has a function of capturing the capture target molecule depending on the specific molecular structure. You may have. For example, it may be a protein or a metal. Specific examples of the capturing body include an antibody and a molecular template polymer.

  The manufacturing method of the molecular template polymer (MIP) 12 is as follows. For example, first, a functional monomer that interacts with the template molecule 10 through an ionic bond or hydrogen bond in the presence of the template molecule 10 that is a molecule to be captured or a similar chemical substance, is used as needed. Polymerize with the ingredients to fix the template molecule 10 within the polymer. At that time, the copolymerization ratio between the functional monomer and the other monomer component varies depending on the type of each monomer component and is not particularly limited. For example, functional monomer: other monomer component = 1: 16 to 1:64 (molar ratio). In particular, functional monomer: other monomer component = 1: 32. Thereafter, the template molecules are removed from the polymer by washing. The cavity (space) remaining in the polymer becomes a recognition site that memorizes the shape of the template molecule 10 and also has a chemical recognition ability by the functional monomer fixed in the molecular template polymer (MIP) 12. .

  As the template molecule 10, for example, a molecule into which a molecule to be captured is derivatized and a functional group that undergoes a copolymerization reaction with a monomer that forms a molecular template polymer may be used. By forming a covalent bond by a copolymerization reaction between the template molecule 10 and the monomer, the interaction between the two becomes stronger, and the fitting property between the template molecule 10 and the monomer is improved. Thereby, as the molecular template polymer (MIP) 12, it is possible to bring about advantageous properties such as improvement in the capture efficiency of the capture target molecule. As a monomer to be copolymerized with such a derivatized capture target molecule, a monomer having two or more functional groups, or a plurality of types, as in the case of a raw material monomer when a non-derivatized capture target molecule is used as the template molecule 10 A combination of these monomers can be used.

  Examples of the functional group that is introduced into the template molecule 10 and that undergoes a copolymerization reaction with the monomer include polymerizable substituents such as an acryloyl group, a methacryloyl group, a vinyl group, and an epoxy group. A methacryloyl group is particularly preferable.

  The molecules to be captured include substances that can exist only in a solid state at normal temperature and pressure, and include chemical substances that can exist as fine particles in gas or liquid. However, those having a corrosive effect, a dissolution effect, a modification effect, etc. with respect to the molecular template polymer are not suitable as target molecules.

  In general, when a molecular template polymer is produced, as described above, a target molecule or its derivative is synthesized as a template molecule, and then the target molecule is captured by removing the template molecule by washing. The recognition site (space) is formed. In the molecular template polymer synthesized as described above, the template molecule may remain in the polymer even after washing. When such a molecular template polymer is used for sample pretreatment, template molecules remaining in the molecular template polymer may elute into the sample after pretreatment and flow into the quantification unit 65 (see FIG. 1). In this case, if the template molecule that has flowed into the quantification unit 65 is a measurement inhibitor that disturbs the test result, the analysis accuracy obtained by the quantification unit 65 decreases.

  Therefore, if the capture target molecule is a measurement inhibitor that disturbs the analysis result even if it remains in the sample in a trace amount, such as phospholipid, the capture target molecule or its derivative is used as a template molecule. It is preferable to prepare a molecular template polymer using a molecule other than.

  For example, when the capture target molecule is a molecule having a phosphate group, the molecular template polymer is preferably synthesized using a molecule different from the capture target molecule as the template molecule. In this case, the template molecule is a molecule that mimics the molecular structure of a molecule having a phosphate group, has a polar group in the molecule, and this polar group has binding sites with other atomic groups on both sides thereof. It can be used.

  For example, when a molecular template polymer for capturing phospholipids is synthesized, it is preferable to use a molecule that mimics the molecular structure of phospholipid without using phospholipid or a derivative thereof as a template molecule. As a molecule that mimics the molecular structure of the phospholipid, a molecule having a ketoprofen skeleton can be used from the viewpoint of the molecular structure, functional group, and the like. For example, S-ketoprofen (S-ketoprofen) can be used.

  Specifically, in the presence of the template molecule S-ketoprofen (S-ketoprofen), the polymerization of vinyl monomer as a functional monomer and other monomer components such as styrene and divinylbenzene as required is initiated. A molecular template polymer can be obtained by copolymerizing the agent together.

  When the vinyl monomer as the functional monomer is copolymerized with another monomer component, the copolymerization ratio varies depending on the monomer component, the type of template molecule, and the like and is not particularly limited.

  The molecular template polymer thus obtained has a recognition site capable of specifically binding to a molecule having a ketoprofen skeleton. As described above, since the ketoprofen skeleton has a molecular structure that mimics the molecular structure of phospholipid, this recognition site functions as a recognition site for phospholipid.

  When S-ketoprofen (S-ketoprofen) is used as a template molecule, derivatization is performed by introducing polymerizable substituents such as acryloyl group, methacryloyl group, vinyl group, epoxy group, etc. into S-ketoprofen (S-ketoprofen). The template molecule may be used as a template molecule.

  The molecular template polymer may be prepared by performing a polymerization reaction in the presence of a template molecule, and the interaction between the template molecule and the surrounding vinyl monomer is not limited to a covalent bond, but an ionic bond, Some or a combination of hydrogen bonds, van der Waals forces, hydrophobic-hydrophobic bonds, etc. may be used.

  3A and 3B are sectional views of the molecular template polymer fine particles 61 accommodated in the pretreatment unit 60. FIG. The molecular template polymer fine particle 61 has a two-layered core-shell type structure in which one shell layer 21 is provided around the core layer 20.

  As the core-shell type molecular template polymer fine particles, for example, as shown in FIG. 3A, a fine particle structure in which a polystyrene layer is used as the core layer 20 and a molecular template polymer layer is formed as the shell layer 21 can be used. These molecularly-templated polymer particles with a core-shell structure are submicron-sized and uniform in particle size, so they are closely packed when arranged in a column or flat form, and are highly recognized for target molecules. Gain power.

  As the shell layer 21, it is only necessary to have a layer of a molecular template polymer that captures the measurement-inhibiting substance as a surface layer outside the core layer 20. In other words, it is only necessary to synthesize particles having, as a surface layer, a molecular template polymer layer that captures a measurement-inhibiting substance.

The molecular template polymer fine particle 61 is not limited to a two-layer core-shell type (see FIG. 3A) having a single shell layer 21 around the core layer 20, but a three-layer core-shell 1- Shell type 2 fine particles can also be used. As shown in FIG. 3B, the core-shell 1-shell 2 type molecular template polymer fine particle 61 having a three-layer structure has a first shell layer 23 around the core layer 22, and a second shell layer 24 around the first shell layer 23. It has a core-shell 1-shell 2 type structure. With this structure, for example, Fe ₂ O ₃ (iron oxide) beads are used as the core layer 22, a polystyrene layer is formed as the first shell layer 23, and a molecular template polymer layer is formed as the second shell layer 24. By forming a molecular template polymer fine particle having magnetism can be prepared.

Here, as the core layer 22 of the molecular template polymer particles 61, the example of using _Fe 2 _{O 3} (iron oxide), the core layer 22, the _Fe 2 _{O 3} (iron oxide) other than the magnetic substance It may be used.

  Thereby, the molecular template polymer fine particle 61 functions as a magnetic substance, and after capturing the measurement inhibiting substance by the surface layer, the molecular template polymer fine particle 61 can be moved by, for example, a magnet, and handling becomes easy. Specifically, for example, a magnetic material having the above-described molecular template polymer on the surface layer is immersed in a specimen containing a measurement inhibitory substance together with a detection target (target molecule) such as serum and urine, and left for a certain period of time. Then, after capturing the measurement-inhibiting substance with the molecular template polymer, the magnetic material can be easily pulled up from the specimen by a magnet or the like. As a result, measurement-inhibiting substances can be separated from substances contained in the specimen.

Among them, the core-shell 1-shell 2 type molecular template polymer fine particles are composed of the molecular layer polymer layer as the second shell layer 24 and the Fe ₂ O _{3 as the} core layer 22 by the polystyrene layer as the first shell layer 23. Adhesion with (iron oxide) beads is enhanced, and a stable molecular trapping function by the molecular template polymer can be obtained.

  A synthesis scheme of the core-shell type molecular template polymer fine particles is shown below. Polymerization reaction is carried out in the presence of fine particles (hereinafter simply referred to as “core beads”) containing the components that form the core layer, and further, centrifugation, hydrolysis, and washing are performed to synthesize polymer beads having a molecular template polymer on the surface. it can.

  That is, it is characterized in that molecular template polymer fine particles can be produced by polymerizing a template molecule and a polymerizable vinyl monomer (functional monomer) in the presence of core beads (fine particles). Thereafter, fine particles of the molecular template polymer can be finally obtained through a centrifugation step, a hydrolysis step, and a washing step.

  In the molecular template polymer fine particles obtained in this way, the core beads (fine particles) are coated with a molecular template polymer synthesized using the raw material monomers of the molecular template polymer and the template molecules (capture target molecules or derivatives thereof). The molecular template polymer that coats the core beads (fine particles) has a recognition site for the molecule to be captured.

  For example, by using polystyrene beads as the core beads and performing the polymerization reaction, centrifugation, hydrolysis, and washing as described above, the core-shell type molecular template polymer fine particles having a two-layer structure shown in FIG. 3A are synthesized. Can do.

Further, for example, by using Fe ₂ O ₃ (iron oxide) beads as the core beads and forming a polystyrene layer on the surface in advance, the polymerization reaction, centrifugation, hydrolysis, and washing are performed as described above. The core-shell 1-shell 2 type molecular template polymer fine particles having the three-layer structure shown in FIG.

  In the chemical analyzer of Example 1 described above, it is possible to perform high-precision analysis because quantitative analysis can be performed after removing measurement-inhibiting substances such as phospholipids that disturb the analysis with a molecular template polymer. Become.

  In addition, in the chemical analysis apparatus of Example 1, by using a molecular template polymer, measurement inhibitory substances can be continuously removed without using a complicated apparatus configuration or a large-scale apparatus configuration. An efficient and high-throughput analysis can be performed.

  FIG. 4 illustrates an example of a processing flow when performing from the pretreatment process to the analysis process using the molecular template polymer (MIP) described above. First, a specimen to be examined is passed through a vial, syringe, column, or the like filled with a molecular template polymer (MIP). At this time, these are stirred and shaken in the presence of the specimen and the molecular template polymer (MIP). As a result, the measurement-inhibiting substance in the sample can be effectively captured by the molecular template polymer (MIP). Next, the supernatant liquid in the suspension obtained by stirring the specimen and the molecular template polymer (MIP) is collected. And the component contained in the fractionated supernatant liquid is analyzed.

  According to the flow described above, for example, measurement-inhibiting substances such as phospholipids are removed from the sample solution, so that highly sensitive analysis of the target molecule becomes possible.

  FIG. 5 shows a schematic configuration of a preprocessing apparatus 400 according to the second embodiment. The pretreatment device 400 can be applied as the pretreatment unit 60 of the chemical analysis device 600 shown in FIG. 1, for example, and includes a syringe-like container 40. The container 40 is filled with a molecular template polymer 41 that captures the phospholipid 43. As the molecular template polymer 41, the same molecular template polymer as described in Example 1 can be used. The specimen 42 includes a phospholipid 43 serving as a measurement inhibitor. Further, the specimen 42 includes molecules 44 and molecules 45 that are detection objects (target molecules).

  First, the specimen 42 is passed through the container 40. The phospholipid 43 in the specimen 42 passed through the container 40 is captured by the molecular template polymer 41 and removed. By analyzing the specimen 42 after being pretreated in this way, it is possible to perform an accurate analysis.

  FIG. 5 shows an example in which the molecular template polymer 41 captures and removes the phospholipid 43 in the specimen. However, the pretreatment apparatus 400 can remove measurement-inhibiting substances other than phospholipids such as phospholipid derivatives, phosphoproteins, phosphopeptides, and the like by appropriately selecting the type of the molecular template polymer 41.

  In Examples 1 to 2 described above, the case where molecular template polymer fine particles are used has been described as an example. However, a pulverized molecular template polymer having a small particle size and a uniform particle size may be used as a capture body for the measurement inhibitor by pulverizing and classifying the powder obtained after the synthesis of the molecular template polymer. . However, in order to increase the sensitivity of detection of the target molecule, it is preferable that the surface area of the molecular template polymer is larger. For this reason, in order to make the particle size of the capturing body smaller, it is preferable to use molecular template polymer fine particles in which the surface of the core bead having a particle size of submicron order is coated with the molecular template polymer as described above.

  Further, according to the above-described Examples 1 to 2, by using molecular template polymer fine particles having a molecular template polymer as a capturing substance for a measurement inhibitor, a separation apparatus (HPLC which has been conventionally required for quantitative analysis) ) And the like can be quickly and quantitatively analyzed without using a high-cost device. For this reason, it can contribute to the cost reduction and size reduction as a whole chemical analyzer. Offline separation may be used.

[Experimental Example 1]
Next, the method for producing the molecular template polymer will be described in more detail. In the following, the case of synthesizing molecular template polymer fine particles for capturing phospholipid, which is a measurement inhibitor that disturbs chemical analysis, according to the above procedure will be described.

(Synthesis of molecular template polymer fine particles)
In Experimental Example 1, a molecular template polymer that captures phospholipids was synthesized using S-ketoprofen as a template molecule.

  Specifically, a pulverized molecular template polymer was synthesized as described below. First, S-ketoprofen (0.10 mmol / L, 259 mg), MAA (0.48 mmol / L, 410 μL), EDMA (1.56 mmol / L, 2.95 μL), 4-styrenesulfonic as template molecules were placed in a vial. Acid (100 mg) and AIBN (17.5 mg) were dissolved in 50 mL of a solvent (acetonitrile: 45 mL, ion-exchanged water: 5 mL). Next, the septum cap was put on and purged with nitrogen, purged with nitrogen for 10 minutes, and then reacted at 70 ° C. for 12 hours.

  The polymerization solution was collected, centrifuged, and the supernatant solution was removed, followed by Soxhlet washing. Specifically, it was washed with methanol / acetic acid = 9: 1 by Soxhlet washing and then with methanol.

  By the above method, a molecular template polymer for removing phospholipid could be prepared. In order to confirm that ketoprofen was sufficiently removed from the molecular template polymer by the above Soxhlet washing, the concentration of ketoprofen was measured by HPLC in the course of Soxhlet washing. The measurement results are shown in Table 1.

[Experimental example 2]
Using the molecular template polymer (MIP) synthesized in Example 1, the ability to remove phospholipids was evaluated. First, as a preliminary experiment, the absorbance at various phospholipid concentrations was measured. Table 2 shows the measured absorbance at each phospholipid concentration. These absorbance measurements were plotted against each phospholipid concentration to create a calibration curve, and the relationship between phospholipid concentration and absorbance was calculated. The calibration curve was prepared by using 1-Palmitoyl-2-oleoyl-sn-glycero-3-PC (1,2-POPC, 1-Palmitoyl-2-oleoyl-sn-glycero-3-Phosphocholine as phospholipid. ) Was used. FIG. 6 shows the relationship between phospholipid concentration and absorbance. As shown in FIG. 6, the absorbance plot for each phospholipid concentration had a linear relationship. When the phospholipid concentration is x and the absorbance is y, these relationships are approximated by a calculation formula of y = 0.0324x + 0.0165, and the correlation coefficient R2 = 0.9894.

  Next, an aqueous phospholipid solution was prepared so that the phospholipid concentration was 1.5 mg / mL, and 1 mL each of this aqueous solution was placed in 5 vials. In addition, the same phospholipid as the phospholipid used for preparation of the calibration curve of FIG. 6 was used. Next, the molecular template polymer (MIP) synthesized in Experimental Example 1 is placed in each vial containing the phospholipid aqueous solution in 10 mg, 20 mg, 30 mg, 40 mg, and 50 mg, respectively, at a temperature of 25 ° C. and 400 rpm for 60 minutes. Stirred and shaken. Thereafter, the obtained suspension was centrifuged, and the absorbance of the supernatant was analyzed using a phospholipid quantification ELISA kit (removal ability evaluation experiment).

  The phospholipid concentration of each supernatant is calculated from each absorbance obtained by the above-described removal ability evaluation experiment, using the calibration curve of FIG. 6, and the phospholipid reduction concentration (phospholipid concentration reduction) and the removal rate are calculated. Calculated. The phospholipid reduction concentration is the difference between the phospholipid concentration after the removal ability evaluation experiment and the initial concentration (1.5 mg / mL). Table 3 shows the phospholipid reduction concentration and the phospholipid removal rate at each molecular template polymer (MIP) concentration. Further, FIG. 7 shows the relationship between the molecular template polymer (MIP) addition concentration and the phospholipid reduction concentration. In FIG. 7, the horizontal axis (x-axis) shows the concentration of molecular template polymer (MIP) added [mg / mL], the first vertical axis shows the reduced concentration of phospholipid [mg / mL], and the second vertical axis The axis indicates the phospholipid removal rate [%] corresponding to the first vertical axis.

  As shown in FIG. 7, the phospholipid reduction concentration, that is, the removal rate of phospholipids increases as the addition amount of molecular template polymer (MIP) increases. With the addition amount of 50 mg, a removal rate of 90% or more is achieved. It was.

DESCRIPTION OF SYMBOLS 10 ... Template molecule, 11 ... Recognition site | part, 12 ... Molecular template polymer, 101 ... Monomer raw material A, 102 ... Monomer raw material B, 103 ... Monomer raw material C, 20, 22 ... Core layer, 21 ... Shell layer, 23 ... 1st Shell layer, 24 ... second shell layer, 400 ... pretreatment device, 40 ... vessel, 41 ... molecular template polymer, 42 ... analyte, 43 ... phospholipid, 44, 45 ... molecule to be detected, 600 ... chemical analysis Device: 60 ... Pretreatment unit, 61 ... Molecular template polymer fine particle, 62 ... Sample introduction unit, 621 ... Channel, 64 ... Switching unit, 65 ... Quantitative unit, 651 ... Sending channel, 66 ... Drain, 661 ... Discharge flow Road

Claims (9)

A pretreatment unit containing a molecular template polymer that captures a molecule having a polar group contained in a specimen;
A chemical analysis apparatus comprising: a quantitative unit that quantifies a component contained in the specimen passed through the pretreatment unit.   The pretreatment unit captures at least one selected from the group consisting of a phospholipid, a phospholipid derivative, a phosphoprotein, and a phosphopeptide as the molecule having the polar group by the molecular template polymer. The chemical analysis apparatus according to claim 1.   The chemical analysis apparatus according to claim 1, wherein the pretreatment unit accommodates molecular template polymer fine particles having the molecular template polymer on a surface thereof.   The chemical analysis apparatus according to claim 1, wherein the pretreatment unit stores a polymer having a recognition site capable of specifically binding to a molecule having a ketoprofen skeleton as the molecular template polymer.   The chemical analyzer according to claim 1, wherein the quantification unit is a mass spectrometer, a liquid chromatograph, or a liquid chromatograph mass spectrometer. A pretreatment device used in a chemical analyzer for quantitative analysis of components contained in a specimen,
The pretreatment apparatus includes a molecular template polymer that captures a molecule having a polar group contained in the specimen, and the molecular template polymer removes the molecule having the polar group from the specimen. Pre-processing device.   The pretreatment device removes at least one selected from the group consisting of a phospholipid, a phospholipid derivative, a phosphoprotein, and a phosphopeptide as the molecule having the polar group by the molecular template polymer. The pretreatment device according to claim 6. The sample is passed through a pretreatment unit containing a molecular template polymer that captures molecules having polar groups,
A chemical analysis method comprising quantitatively analyzing a component contained in the specimen passed through the pretreatment unit.   As a molecule having the polar group, a sample is passed through the pretreatment unit containing the molecular template polymer that captures at least one selected from the group consisting of phospholipids, phospholipid derivatives, phosphoproteins, and phosphopeptides. The chemical analysis method according to claim 8.

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